Duped by dumping syndrome: non-endemic Vibrio cholerae bacteremia in an immunocompetent host with gastric bypass surgery, a case report

Extra-intestinal infection with non-O1/non-O139 strains of Vibrio cholerae (NOVC) is rare, though bacteremia and hepatobiliary manifestations have been reported. Reduced stomach acid, or hypochlorhydria, can increase risk of V. cholerae infection. We describe a 42-year-old woman with hypochlorhydria due to untreated Helicobacter pylori infection, gastric-bypass surgery, and chronic proton pump inhibitors (PPI) exposure, who developed acute diarrhoea following raw oyster consumption. Her symptoms were attributed to rapid gastric emptying (dumping syndrome) after a negative limited stool work-up. She had persistent diarrhoea, weight loss, and after 5 months was admitted with acute cholecystitis and NOVC bacteremia, requiring cholecystectomy. This is the first reported case of NOVC bacteremia and cholecystitis in a patient with gastric bypass. This case highlights the potential for NOVC biliary carriage, the role of hypochlorhydria as a risk factor for Vibrio infection, and the importance of excluding infectious diarrhoea in patients with new onset of symptoms compatible with dumping syndrome and a relevant travel history.


BACKGROUND
Epidemic strains of Vibrio cholerae, including the O1 and O139 subtypes, are typically associated with poor drinking water access and suboptimal sanitation.These strains encode the ctx (cholera toxin) and tcpA (toxin-coregulated pilus) genes which are known to cause severe acute diarrhoea and volume depletion [1].Over 200 serotypes of V. cholerae have been identified.Non-O1/non-O139 strains (NOVC) have been increasingly reported in clinical and environmental settings.V. cholerae are found in marine environments including water, sediment, abiotic, and biotic surfaces (e.g.crustaceans exoskeleton) [2].Growth and survival is optimized in warm, low salinity water.Rising global temperatures and severe precipitation events leading to reduced salinity of ocean waters, have potentiated the global burden of NOVC infection and has led to significant geographic expansion over the last 40 years [3,4].Clinical isolates of NOVC have been linked to diarrhoeal illness, wound infection, gastroenteritis, and bacteremia [5][6][7].Infection is frequently associated with consumption of seafood (primarily crustaceans and molluscs) [8][9][10] or soft tissue injuries exposed to seawater [11], primarily in immunocompromised hosts [5,12].

CASE PRESENTATION
A 42-year-old Hispanic female with a history of Roux-en-Y gastric bypass surgery in early 2021 resulting in 110 pound weight loss and incompletely treated H. pylori infection (diagnosed in 2018) on chronic omeprazole (>3 years), first presented to a Boston-based clinic in October of 2021 with acute onset diarrhoea and abdominal pain.She had just travelled to Miami, Florida and then Santo Domingo, Dominican Republic, where she reported eating raw oysters (See Fig. 1.Timeline).A few days after her oyster exposure, she developed profuse explosive watery diarrhoea and near syncope.She had a limited initial workup including Salmonella/Shigella culture, Campylobacter Enzyme Immunoassay (EIA), and Shiga toxin EIA, all of which were negative.H. pylori stool antigen was positive.She continued her baseline omeprazole and was treated with amoxicillin and clarithromycin for 2 weeks.Persistent diarrhoea and ongoing weight loss was attributed to dumping syndrome typical of patients with a history of gastric bypass.Subsequent H. pylori antigen testing was negative.
In January of 2022, she was evaluated for an episode of acute cholecystitis.Abdominal ultrasound showed a distended gall-bladder with cholelithiasis and positive sonographic murphy sign.No blood cultures or stool studies were sent.She was discharged  with a ten-day course of amoxicillin/clavulanic-acid.She continued to have stable watery, loose stools.In March of 2022 she travelled to Texas, but reported no consumption of seafood or water exposures.A few days after her return she again developed worsening abdominal pain, now with vomiting.At this juncture she had lost an additional 30 pounds.Labs were notable for elevated liver enzymes: aspartate aminotransferase 601 IU/L (ref 9-32 IU/L), alanine aminotransferase 349 IU/L (ref 4-37 IU/L), alkaline phosphatase 427 IU/L (ref 40-129 IU/L), total bilirubin 1.2 mg dL −1 (ref 0.1 to 1.2 mg dL −1 ), direct bilirubin 0.8 mg dL −1 (ref 0.1-0.3mg dL −1 ).Clostridium difficile antigen was negative.Abdominal ultrasound showed cholelithiasis and MRCP was consistent with cholecystitis.Two sets of blood cultures were positive from both aerobic and anaerobic bottles for Gram-negative rods.
Blood culture Gram-stain demonstrated curved Gram-negative rods (Fig. 2a).Beta hemolysis was observed on sheep blood agar (though the colonies appeared confluent) (Fig. 2b).The isolate was oxidase positive.On MacConkey agar, it appeared non-lactose fermenting (Fig. 2c).It was further subbed to thiosulphate citrate bile salt sucrose agar (TCBS) which demonstrated yellow colonies (Fig. 2c).The isolate was confirmed as V. cholerae by MALDI-TOF using the VITEK MS (bioMérieux, France) and then later by the Centres for Disease Control and Prevention (Atlanta, GA) as a member of the nontoxigenic serotype (non-O1, non-O139).The ctxA toxigenic marker was negative by multiplex PCR.Stool cultures on antibiotics were negative including on Vibrio sp.selective media.The isolate was susceptible to ampicillin, ciprofloxacin, levofloxacin, tetracycline, and trimethoprim-sulfamethoxazole.Her liver function tests improved and she was discharged to complete a 10 day course of ciprofloxacin.Her weight, appetite, and stool consistency improved slowly over the subsequent 2-3 months.
The organism was confirmed to be V. cholerae (non-O1/O139), MLST 213 [14] The length of the draft assembled genome was four million base pairs with a N50 of 286 315.No antibiotic resistance genes were identified using ResFinder [15].It was not associated with any known clusters using the NCBI Pathogen Detection tool (SAMN27723935) [16] or cases of known human infection.Virulence genes detected using the VFDB database tool [17] included those associated with adherence, anti-phagocytosis, chemotaxis, iron uptake, type VI secretion systems, toxin formation (non-ctx-AB or tcpA), biofilm formation, endotoxin, fibrial adherence, and immune evasion (see Table 1).

DISCUSSION AND CONCLUSIONS
NOVC infection is typically acquired by ingestion of infected seafood or wound exposure to marine environments and can result in severe clinical manifestations.Unlike epidemic strains, NOVC isolates typically do not carry the ctxAB and cpA genes.Despite this, NOVC strains are associated with significant genetic diversity and may harbour other virulence factors acquired via horizontal gene transfer.The toxigenic potential of NOVC strains have been attributed to genes coding for type III and type VI secretion systems, enterotoxins, and hemagglutinin proteases [18,19].This isolate demonstrated genes coding for a type VI secretion system, associated with bacterial persistence [20], and an hlyA gene, coding for hemolysin, associated with gut colonization and immune evasion, and gastroenteritis in NOVC strains [18,21].
Table 1.Virulence factors detected by whole genome sequencing using the VFDB database [17].Of note, no antibiotic resistance genes were identified using ResFinder [15] Virulence addition to pathogen virulence factors, host susceptibility to infection is necessary to result in clinical disease.Reduced stomach acid has been proposed as a possible risk factor for both epidemic and non-epidemic V. cholerae infection due to the loss of protection conferred by normal gastric acid levels [22][23][24][25][26].Our patient was on chronic omeprazole and also had Roux-en-Y gastric bypass surgery which has been associated with decreased basal and peak production of gastric acid [27] She also had a history of untreated H. pylori which has been associated with an increased risk of cholera infection [28], possibly due to the induction of hypochlorhydria [29,30].
NOVC presenting as acute biliary disease has been described in the literature in numerous case reports from both epidemic [31,32] and non-epidemic strains [33,34].In one retrospective review, Chen et al. reported that among 83 NOVC infections reported from Taiwan (between 2009 and 2014), 12 patients (14.5 %) presented with biliary tract infection [35].Biliary carriage of V. cholerae has also been suggested, though little has been published describing this phenomenon.In 1967, O1 V. cholerae was isolated from duodenal aspiration of two convalescent cholera patients in their post-infection period (7 days or more after their last positive stool culture) after stimulation with cholecystokinin, suggesting the gallbladder as a reservoir [36].Also in 1967, a patient later named 'Cholera Dolores' was identified as a probable long term carrier of V. cholerae.She continued to shed morphologically consistent V. cholerae in the stool from 1962 to 1966.She had ongoing symptoms of obstructive biliary disease.Duodenal fluid aspiration after a fatty meal clearly indicated the high presence of V. cholerae [37].The persistence of V. cholerae in the biliary tract may be due to the production of biofilms in the presence of bile [38], which may form around gallstones.In our patient, subsequent bacteremic events required the formation of biofilms to allow for bacterial persistence over the course of months.Intestinal and biliary biofilm formation by V. cholerae is an important precursor to systemic disease [2].
Treatment of invasive NOVC infection typically requires systemic antibiotics (e.g.ampicillin, piperacillin-tazobactam, ceftriaxone, cefepime, ciprofloxacin, or carbapenems).Drug resistance in NOVC strains has been rising steadily, with high rates of ampicillin resistance (up to 88 % in some settings) and, though rare, increasing reports of ciprofloxacin resistance word-wide (from 3 % by 2010-7 % in 2020) [39].Susceptibility on extra-intestinal isolates should be performed, as drug resistance will impact antibiotic selection.
To our knowledge this is the first reported case of NOVC cholecystitis and bacteremia in a patient with prior gastric bypass surgery.Though not immunocompromised, this patient's primary risk factor for systemic infection was her recent gastric bypass procedure and the resultant hypochlorhydria.H. pylori infection and PPI use may have also played a role.Her most likely exposure (oyster consumption) and compatible clinical history was 5 months prior to developing bacteremia, though we cannot exclude the possibility of a more recent exposure.While her persistent diarrhoea may have been related to dumping syndrome, any superimposed infectious diarrhoea was missed due to a limited stool workup.V. cholerae would not have been detected from the stool workup performed and further stool studies were not pursued until after the diagnosis of V. cholerae was made.She did receive antibiotic therapy for H. pylori and the first episode of cholecystitis that were active against V. cholerae.It is possible that she entered into a carrier state following her initial infection, and/or that antibiotics were poorly absorbed due to malabsorption.In other biliary infections, such as with Salmonella typhi, biliary carriage is not easily eradicated with antibiotics due to biofilm formation on gallstones [40].This case highlights the need for appropriate microbiological testing based on patients' exposure history and the clinical importance of excluding infectious diarrhoea in patients with new onset of symptoms or ongoing symptoms compatible with dumping syndrome and a relevant travel history.Clinical microbiology labs may offer Vibrio testing through expanded gastrointestinal multiplex PCR panels or through clinician-directed requests for Vibrio cultures.Clinicians should be aware of their institutional protocols for stool culture workup, as Vibrio is often not routinely assessed.We highlight gastric bypass surgery as a risk factor for V. cholerae infection.Further studies are needed to understand the role of gallbladder carriage in V. cholerae.Comments: 1. Description of the case(s) This work describes an interesting case with novel interest.The authors have covered the history, presentation, investigation, and management of the case thoroughly but concisely, maintaining a clear narrative throughout, supported by a timeline of key dates and exposures.2. Presentation of results WGS results were well described, covering the specifics of the sequencing and bioinformatics done whilst remaining accessible to non-specialist readers.The table summarising the key gene virulence factors identified was particularly helpful to a non-Vibrio expert.3. How the style and organization of the paper communicates and represents key findings The paper is well structured into clear sections, and the figures, particularly the timeline and the summary of the functions of key genes identified by sequencing, support and enhance the narrative.4. Literature analysis or discussion The discussion nicely frames the case in the context of the literature, and makes clear which elements of this case are novel.All potential questions I had whilst reading through the case description have been considered by the authors, and they have summarised with good take-home learning points.5. Any other relevant comments A very enjoyable, well-written read, describing an interesting case with clearly laid out novel interest.The authors have covered the specifics of this case and the follow-up sequencing work in sufficient detail to make it clear to the reader exactly what was done, without getting lost in unnecessary details which could disrupt the narrative.It also raised excellent considerations on how the presentation and epidemiological history of V. cholerae infection may not fit the paradigm.Well done and thank you to the authors for this interesting piece of work.

Please rate the quality of the presentation and structure of the manuscript Very good
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes

Fig. 2 .
Fig. 2. A. Gram-stain of cultured organism demonstrating curved Gram-negative rods.b.Colonies on sheep blood agar.Colonies appear to be betahemolytic when confluent.Beta hemolysis is less apparent on sheep agar (bottom) when colonies are spread further apart.c.Lactose non-fermenting organism demonstrated (top).d.Yellow colonies on thiosulphate citrate bile salt sucrose agar (TCBS).

Author response to reviewers to Version 1
Duped by dumping syndrome: Non-Endemic Vibrio choleraebacteremia in an immunocompetent host with gastric bypass surgery, a case reportReviewer comments:Please rate the quality of the presentation and structure of the manuscript Reviewer 1: Good To what extent are the conclusions supported by the data?Reviewer 1: Strongly support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 1: No: If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Reviewer 1: Yes: Reviewer 1 Comments to Author: Dear author, A well written case report case report highlighting the effect of gastric-bypass surgery on Vibrio cholera.Some suggestions to optimise the report.Background • Could you explain more in detail the increasing Non-01/non-0139 strains(NOVC) in the clinical setting.You only strength the potentially influence to climate changes in temperature.o We elaborated on how increased temperature and lower salinity oceans can potentiate the growth of NOVC, to add more context about the influence of climate change • Do you have more literature for the association of NOVC with consumption of seafood or soft tissue injuries exposed to seawater o I added some studies demonstrating the association with seafood (PMID 7304565, 7235397, 6490304 and sea water exposure to wounds ( 35567650) Case presentation You state chronic omeprazole use -could you more in detail define the time period.o Noted greater than 3 years, added to the text • How you define the dumping syndrome?o I adjusted the wording to indicate that this is rapid gastric emptying in the abstract • I suggest to add the reference values for the elevated liver enzymes o Added reference values Discussion and conclusion • Given the absence of standard therapeutic guidelines for NOVC infections, antimicrobial susceptibility testing remains critical.o Susceptibility testing results were entered • You should describe more in detail the antibiotic testing/treatment.o A short paragraph was added regarding AST testing/treatment (limited by space for further elaboration) • Was the H. pylori infection eradicated?o I now indicated that h pylori testing was negative after her second round of treatment • I suggest to stress more on the relation of consumption of raw oyster consumption in stead of chronic omeprazole treatment.o I reiterated the point that she had consumed raw oysters (epi exposure) and the discussion o The discussion of omeprazole is in relationship to host factors that may potentiate invasive infection, but a proper exposure is required in order to have an infection at all -I indicated that a possible risk factor for "invasive" infection was related to gastric PH but reiterated the need for an epidemiological exposure o I removed one sentence about stomach acid and vibrio reproduction (to allow space to expand on exposure risk) • I suggest to add the publication of De Keukeleire et al., 2018 -atypical manifestation of Vibrio cholerae: fear the water -a case report from an epidemic NOVC strain.o I added this citation to the manuscript • You should stress more the relation of production of biofilms in order to develop bacteremia.o I added a line to the abstract about potential for biliary carriage, i added a sentence describing the need for biofilm formation to develop systemic infection Please rate the quality of the presentation and structure of the manuscript Reviewer 2: Very good To what extent are the conclusions supported by the data?Reviewer 2: Strongly support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 2: No: If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Reviewer 2: Yes: VERSION 1 Editor recommendation and comments https://doi.org/10.1099/acmi.0.000517.v1.5 © 2023 Bosworth A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Bosworth; Public Health England, UNITED KINGDOM Date report received: 29 September 2023 Recommendation: Minor Amendment Comments: This is a study that would be of interest to the field and community.The reviewers have highlighted minor concerns with the work presented.Please ensure that you address their comments.Reviewer 2 recommendation and comments https://doi.org/10.1099/acmi.0.000517.v1.3 © 2023 Kettles R.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Rachel Kettles; UNITED KINGDOM https://orcid.org/0000-0002-5968-7930Date report received: 12 September 2023 Recommendation: Accept

factor Associated genes detected
linked with climate change: a neglected research field?Environ Microbiol 2020;22:4342-4355.4. Christaki E, Dimitriou P, Pantavou K, Nikolopoulos GK.The impact of climate change on cholera: a review on the global status and future challenges.Atmosphere 2020;11:449.The work presented is clear and the arguments well formed.This study would be a valuable contribution to the existing literature.